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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:781-794 (2006)
© 2006 The Gerontological Society of America

Calorie Restriction in Mice: Effects on Body Composition, Daily Activity, Metabolic Rate, Mitochondrial Reactive Oxygen Species Production, and Membrane Fatty Acid Composition

Sally C. Faulks, Nigel Turner, Paul L. Else and A. J. Hulbert

1 Metabolic Research Centre, and 2 School of Biological Sciences, 3 Department of Biomedical Sciences, University of Wollongong, New South Wales, Australia.

Address correspondence to A. J. Hulbert, PhD, DSc, School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia. E-mail: hulbert{at}uow.edu.au


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Different levels of calorie restriction (CR) (125, 85, 50, or 40 kcal/wk for 1, 3, and 6 months) were examined in mice by using the paradigm of Weindruch and colleagues. Lean and total body mass increased on 125 and 85 kcal/wk, but there was negligible growth on low-energy intake. There was no CR-induced reduction in either daily activity or mass-specific metabolic rate. There was no CR-effect on in vitro reactive oxygen species production by liver or muscle mitochondria at 3 months, but after 6 months the effect was significantly reduced in liver mitochondria from 40 kcal/wk mice compared to 125 kcal/wk mice. Changes in the fatty acid composition of phospholipids from liver, kidneys, heart, brain, and skeletal muscle were observed following 1 month of CR.

DIETARY calorie restriction (CR) remains the only physiological treatment known to slow aging and extend life span in a wide range of animal species (1). It has been most studied in rats and mice, but has also been demonstrated in yeast and several invertebrate species, including Drosophila melanogaster. It is not yet completely clear how CR exerts its effects, although a decrease in oxidative damage is a common finding (1). In rodents, the degree of life-span extension is linearly related to both the degree of CR (to at least 30%–35% of ad libitum intake) and the time for which CR is imposed (2). It has been proposed that CR in rodents extends life span by both slowing the intrinsic rate of aging and suppressing pathogenesis and thus reducing the short-term risk of death (3). Recently, nutritional-shift experiments in D. melanogaster that examined effects on age-specific mortality rates showed changes within 2 days of a shift between full diets and restricted diets (4). These experiments demonstrated that the effects of CR in fruit flies are very rapid, that CR in fruit flies extends life span by reducing the short-term risk of death. They showed that the effects of CR on age-specific mortality rates were rapidly reversible. Surprisingly, they showed no evidence for an influence of accumulated damage in determining life span. Such measurements of rates of age-specific mortality require the use of large numbers of animals, and no similar experiments in rodents have been reported. However, rapid and reversible effects of CR have also been reported in mice (5,6).

CR in rodents decreases oxidative damage to many cellular macromolecules including lipids, proteins, and nucleic acids; however, significant differences are often not observed until many months after the initiation of CR (1). Several years ago, Laganiere and Yu (7) reported that one of the effects of CR in rats is to alter the fatty acid composition of subcellular membranes of liver such that they become more resistant to lipid peroxidation. This observation was later confirmed for rat liver (8), heart (9), and skeletal muscle (10); however, whether it represents a general effect of CR is controversial, as others have either not observed such changes in heart (11) or liver (12) or only observed them after long-term CR (13). In view of the proposal that membrane alteration is the basis of the protective effects of CR (14) and that variation in membrane fatty acid composition is an important factor in the determination of life span (15,16), we have reinvestigated the situation in mice.

We were interested in how widespread any changes in membrane fatty acid composition might be following CR, as well as the relative time course of any such membrane composition changes relative to other effects of CR. We have repeated the CR paradigm used by Weindruch and colleagues (17), except that we have used a different strain of mice. Those researchers used weanling C3B10RF1 mice, which were an F1 hybrid strain from female C3H.SW/Sn inbred mice and male C57BL10.RIII/Sn inbred mice (17). CR has been found to increase the longevity of "many different stocks and strains of mice" (1), although there are two reports of CR decreasing the longevity of some mice strains. Harrison and Archer (18) report this for C57BL/6J inbred mice; however, other researchers report that CR extends longevity in this strain [e.g., (19)]. A small reduction in longevity during CR has also been reported for DBA/2 inbred mice (19). It is of interest that, compared to other mice strains, this strain has been recently reported to show hypersecretion of insulin in response to glucose and a different response to a high-fat diet (20). Because we were not able to obtain the strain originally used by Weindruch and colleagues (17), we chose to examine weanling Quackenbush Swiss (QS) mice available locally to us. This is a robust outbred albino strain of mice originally selected from Swiss mice colonies but, to our knowledge, there are no previous studies on the effects of CR on the longevity of these mice.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals and Animal Care
Weanling male QS mice were purchased from Gore Hill Laboratories (Sydney, Australia). Mice were housed individually in the same room in plastic cages (36 cm x 24 cm x 20 cm) at 22°C and with a 12-hour light/dark cycle. In all cages were wood shavings and a small plastic tube (45 mm diameter x 100 mm long). Following weaning and arrival at the University of Wollongong, all mice were initially fed the 125 kcal/wk diet, and after 1 week they began their respective experimental diet in a staggered manner such that all mice at the time of death had been on their respective experimental diet for 1, 3, or 6 months. Each mouse was fed a precise amount of food daily (at approximately 9:00–10:00 AM) that corresponded to either 125, 85, 50, or 40 kcal/wk. Mice ate all their daily rations. The mice on 125 kcal/wk consumed all the provided food, with very rare occurrences of very small portions being left uneaten. All measurements were made on four mice per day (one from each diet group).

The diets were the same as those described by Weindruch and colleagues (17), except that brewer's yeast and zinc oxide were omitted (due to the use of different vitamin and mineral mixes), and gelatin was added for structural stability. Diet 1 (for the 125 kcal/wk and 85 kcal/wk mice) consisted of (in g/kg dry) casein (152 g/kg), cornstarch (261 g/kg), sucrose (261 g/kg), corn oil (135 g/kg), fiber (56 g/kg), mineral mix (61 g/kg), vitamin mix (24 g/kg), and gelatin (50 g/kg). Diet 2 (for the 50 kcal/wk and 40 kcal/wk mice) consisted of (in g/kg) casein (303 g/kg), cornstarch (158 g/kg), sucrose (158 g/kg), corn oil (135 g/kg), fiber (40 g/kg), mineral mix (112 g/kg), vitamin mix (44 g/kg), and gelatin (50 g/kg). For each of the two diets described, an equal amount of water was added during mixing, and the diet was measured into different daily proportions while heated in a 75°C water bath. The food was measured into small plastic weigh trays as individual daily portions, and was frozen until needed. The diet portions were made up as required, but were not stored frozen for longer than 2 weeks.

Daily Activity
Two days before each mouse was killed, its cage (with the mouse) was placed under an infrared activity monitor (PIR-9038 Passive Infrared Mini Detector; Dick Smith Electronics, Wollongong, Australia) for 24 hours. Four such monitors were simultaneously used to record the daily activity of a single mouse from each experimental group via a PowerLab system (ADInstruments, Sydney, Australia) and a Macintosh PowerBook laptop computer. The infrared detector was able to record any movement in any part of the cage every 2 seconds.

Resting Metabolic Rate
On the day before each mouse was killed, all mice had their resting oxygen consumption measured at an ambient temperature of 22°C. Each mouse was placed in a glass metabolism chamber, and dry CO2-free air was passed through the chamber at ~150 mL/min. The air flow was measured with a Tylan mass flow controller (model FC2805; Billerica MA), and the oxygen concentration of both inlet and dry CO2-free outlet air was measured with an Oxzilla oxygen analyzer (Sable Systems, Henderson, NV). Outputs from both the flowmeter and oxygen analyzer were continuously recorded using a PowerLab system connected to a Macintosh computer.

Body Composition
All mice were weighed and killed by an intraperitoneal injection of sodium pentobarbitone (100 mg/kg). After the mouse was anesthetized, its thorax was opened and a small blood sample was rapidly taken from the heart and placed in a HemoCue B-Haemoglobin Analyzer (Angelholm, Sweden) for measurement of blood hemoglobin. The heart, liver, kidneys, brain, and skeletal muscle (from the hind limb) were rapidly removed and weighed. Most of the liver and skeletal muscle were placed on ice, and mitochondrial preparation began (see below). A small piece of liver and skeletal muscle together with the kidneys, heart, and brain were rapidly frozen in liquid nitrogen for subsequent analysis of membrane fatty acid composition. The testes were removed and weighed as was the skin and fur. The testes and skin and fur were returned to the remainder of the carcass, and this was then frozen (at –15°C) in a sealed container. Later, the frozen carcass of the mouse was placed in a weighed cellulose extraction thimble (28 mm x 80 mm; Whatman, Maidstone, U.K.), then freeze-dried using a Dynavac FD3 freeze drier (Sydney, Australia) at approximately –40°C and a Dynavac Edwards RV-3 vacuum pump. Freeze-drying was performed until a constant mass was reached. Neutral lipid was then extracted using petroleum ether (boiling point = 40°–60°C) in a Soxhlet apparatus. Trial measurements showed that it took 5 hours for fat extraction, so all samples were left for at least 7 hours to ensure that complete extraction occurred. Lean body mass was calculated as total body mass minus the loss of mass during Soxhlet extraction. The liver, kidney, heart, brain, and skeletal muscle samples removed from the mice were included in this calculation and were assumed to be 80% water and to lack neutral lipid.

Mitochondrial Isolation and Reactive Oxygen Species Measurement
Liver samples were chopped coarsely with scissors, and muscle tissue was finely chopped with a single-edge razor blade. They were rinsed and resuspended in 10 volumes of ice-cold medium containing 250 mM sucrose, 5 mM Tris–HCl, 2 mM EGTA, and 1% wt/vol bovine serum albumin (BSA), pH 7.4 at 4°C, then homogenized using six passes of a motorized Teflon/glass homogenizer. The homogenates were centrifuged at 700 g for 10 minutes in a Beckman centrifuge, the supernatant was centrifuged at 10,000 g for 10 minutes, then the pellet was resuspended, respun at 10,000 g, resuspended in isolation medium, and kept on ice. The protein content of each mitochondrial preparation was determined by the method of Lowry and colleagues (21) using BSA standards and absorbance (at 750 nm) measured using a Cary WinUV spectrophotometer (Varian, Melbourne, Australia).

Mitochondrial production of reactive oxygen species (ROS) was determined using the method of Barja (22). This method measures production of superoxide and hydrogen peroxide in that excess superoxide dismutase enzyme (SOD) converts superoxide produced by the mitochondria to H2O2 which, together with the H2O2 endogenously produced by the mitochondria, is then converted to water by excess horseradish peroxidase enzyme (HRP), in turn producing a fluorescent product from homovanillic acid (HVA). The ROS reaction medium consisted of 120 mM KCl, 5 mM KH2PO4, 3 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 0.3% wt/vol BSA, 0.1 mM HVA (pH 7.2, 20°C) with 50 IU SOD/mL, and 12 IU HRP/mL. All chemicals and enzymes were obtained from Sigma Chemical Co. (St. Louis, MO). ROS reaction medium and mitochondria were added to a stirred, temperature-controlled, 1 cm light path quartz fluorescence cuvette in a Hitachi F-4500 Fluorescence Spectrophotometer (excitation wavelength = 312 nm, emission wavelength = 420 nm; San Jose, CA) and allowed 3 minutes for temperature equilibration to 37°C. Following this period, mitochondrial respiration and ROS production were initiated by addition of pyruvate and malate (to a final concentration of 2.5 mM), and the fluorimeter output was continuously recorded. After a stable slope had been obtained (normally 5–10 minutes), the system was calibrated by 2–3 injections of a known amount (0.2 nmol) of a H2O2 standard solution. Mitochondrial ROS production was calculated as picomoles of H2O2 per milligram of mitochondrial protein per minute.

Membrane Fatty Acid Composition
Total lipid was extracted from both frozen tissues and mitochondrial preparations by standard methods (23) using ultrapure grade chloroform/methanol (2:1, vol/vol) containing butylated hydroxytoluene (0.01% wt/vol) as an antioxidant. Phospholipids were separated from neutral lipids by solid-phase extraction on Sep-Pak silica columns (Waters, Milford, MA). Phospholipid fractions were transmethylated (24) and fatty acid methyl esters were separated by gas-liquid chromatography on a Shimadzu 17A gas chromatograph (Shimadzu, Sydney, Australia) with a Restek FAMEWAX capillary column (Bellefonte, PA). Individual fatty acids were identified by comparing each peak's retention time to those of external standards, and were expressed as the mole percentage of total fatty acids. Unsaturation indices (UI; the number of double bonds per 100 acyl chains) were calculated as UI = (% monoenoics) + (2 x % dienoics) + (3 x % trienoics) + (4 x % tetraenoics) + (5 x % pentaenoics) + (6 x % hexaenoics).

The carbon atoms in fatty acyl chains most susceptible to oxidative attack are those between the double-bonded carbons found in polyunsaturates (i.e., the bis-allylic carbons between the –C=C– units) (35). Holman (25) has empirically determined the relative rates of oxidation of different acyl chains, and these values have been used (together with the acyl composition) to calculate a peroxidation index (PI) for each phospholipid extract. PI values were calculated as (0.025 x % monoenoics) + (1 x % dienoics) + (2 x % trienoics) + (4 x % tetraenoics) + (6 x % pentaenoics) + (8 x % hexaenoics).

Statistics
Significant effects were determined by analyses of variance conducted using the JMP IN 3.2.1 statistical package (SAS Institute Inc., Cary, NC). Where analyses of variance revealed a significant effect, Tukey's post hoc test was used to identify significant differences. Significance was set at a level of p <.05, and all values are presented as means ± standard error of the mean excepted where stated.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Growth and Body Composition
The influence of dietary energy intake on body mass and body composition of the mice is shown in Figure 1. In mice, postweaning is a period of rapid growth, and there were significant effects on body mass within the first month on the different energy intakes. After 1 month on their respective diets, the 125 kcal/wk mice had a total body mass that was 231% that of the 40 kcal/wk mice. All groups showed an increase in body mass over the next 5 months; however, this increase was statistically significant for only the 125 and 85 kcal/wk mice. After 6 months, the 125 kcal/wk mice had a body mass that was 311% that of the 40 kcal/wk mice (Figure 1a).


Figure 01
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  		Figure 1. Effect of four levels of energy intake (125, 85, 50, and 40 kcal/wk) on body mass and body composition of male mice. Error bars represent ± standard error of the mean (where they are not observable in the figure, they are smaller than the size of the symbol), N = 6 for each group. a, total body mass; b, lean body mass. Bottom six graphs show masses of liver (c), heart (d), kidney (e), brain (f), skin and fur (g), and testes (h). Tissues masses are presented as percentage of lean body mass

 
Dietary energy intake affected both lean body mass (Figure 1b) and body fat content. After 1 month on their respective diets, the 125 kcal/wk mice had a lean body mass that was 183% that of the 40 kcal/wk mice. After 6 months, they had a lean body mass that was 218% that of the 40 kcal/wk mice. As expected, the greatest difference between the mice was in body fat. After 1 month, the mice had a body fat content that was 21%, 13%, 0.7%, and 0.4% of total body mass (for the 125, 85, 50, and 40 kcal/wk groups, respectively). All groups showed an increase in body fat content over the following 5 months, although this increase was statistically significant for only the 125 and 85 kcal/wk mice. After 6 months on their diets, body fat represented 32%, 24%, 6.5%, and 3.4% of total body mass for the 125, 85, 50, and 40 kcal/wk groups, respectively. Similarly, all groups showed an increase in lean body mass between 1 and 6 months on their different diets. The increase in lean body mass from 1 to 6 months was 23.4, 12.3, 5.1, and 2.3 g, respectively, for the 125, 85, 50, and 40 kcal/wk groups. As a percentage of the 1-month lean body mass, this finding represents a 57%, 40%, 29%, and 13% increase, respectively.

Although lean body mass differed with dietary energy intake and increased with time (especially for the mice on high energy intakes), the liver, heart, and kidney remained a relatively constant percentage of lean body mass irrespective of the actual lean body mass (Figure 1c–e). The liver averaged 5.5% of lean body mass in all groups (range 4.9%–6.2%), the kidney averaged 1.6% of lean body mass in all groups (range 1.4%–1.7%), and the heart averaged 0.63% of lean body mass in all groups (range 0.56%–0.72%).

Unlike these three organs, there was substantial variation in relative size of the brain, skin, and testes (as % of lean body mass) among the different groups (Figure 1f–h). Brain size averaged 1.8% of lean body mass in all groups, but there was considerable variation among the different groups (range 1.2%–2.5%). The relative brain size decreased both with increasing energy intake and over time for each dietary intake. These patterns were due to the fact that the brain had essentially finished its growth before commencement of the treatments and did not greatly change in absolute mass during the 6 months of the experiment. The difference between the dietary energy groups was manifest after 1 month on their respective diets, and changed little in the following 5 months. Absolute brain mass averaged 0.46 g for all groups and ranged from 0.48 g to 0.43 g after 1 month (125 vs 40 kcal/wk mice) and from 0.52 g to 0.41 g after 6 months (125 vs 40 kcal/wk mice).

The skin (and fur) showed the greatest variation in absolute mass of all tissues measured, varying from 2.6 g (in 1 month 40 kcal/wk mice) to 10.0 g (in 6 month 125 kcal/wk mice). From superficial observation, this variation in skin mass appeared to be due to reduced fur thickness, reduced skin thickness, and reduced subcutaneous fat with low energy intake. Skin was the only tissue measured which showed a significant decrease as a relative proportion of lean body mass with energy intake but stayed relatively constant with time (Figure 1g).

In both the 125 and 85 kcal/wk mice, the testes were similar in absolute mass (~0.32 g) and changed little in absolute mass between 1 and 6 months. Consequently, comparing these two groups, the testes represented a greater percentage of lean body mass in the 85 kcal/wk mice compared to the 125 kcal/wk mice (see Figure 1h). In both groups of low energy intake mice at 1 month the testes averaged ~0.13 g in mass (which was about 42% the average testes size of the two high energy intake groups), and in these low energy mice the testes mass was a lower percentage of lean body mass. In the 50 kcal/wk mice, by 3 months the testes had increased in size by 85%, compared to a more modest 26% increase in the 40 kcal/wk mice. Expressed relative to lean body mass, the testes size was greatest in the 50 kcal/wk mice after 3 months on this diet. The testes showed the greatest variability in relative size of all the measured tissues (see Figure 1).

Hemoglobin concentrations of blood are presented in Table 1. In the mice fed the highest energy (125 kcal/wk), blood hemoglobin levels were constant throughout the 6 months of the study. All other groups showed essentially the same pattern, with a small (statistically insignificant) decrease in blood hemoglobin levels following 1 month on their respective diets, but by 3 months, blood hemoglobin had returned to prediet levels where it remained for the remaining 3 months of the experiment. At the end of the study there was no difference in blood hemoglobin levels between the dietary groups.


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  		Table 1. Effect of Calorie Restriction in Mice on Blood Hemoglobin Concentration.

 
Daily Activity and Metabolic Rate
The 24-hour movement of each mouse in its own cage was measured using infrared sensors 2 days before it was killed for biochemical analysis. There was no obvious difference in the diurnal pattern of these movements between treatments, and results are expressed in Figure 2a as percentage of time active. As can be seen from this figure, mice fed 125 kcal/wk exhibited a relatively constant level of daily activity throughout the experiment. The 50 kcal/wk mice exhibited a significant (p <.05) increase in daily activity after 1 month of CR, but this increase declined over the following months such that after 6 months of CR there was no significant difference in activity between any of the dietary groups.


Figure 02
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  		Figure 2. Effect of four levels of energy intake (125, 85, 50, and 40 kcal/wk) on 24-hour activity (a), metabolic rate (MR; resting oxygen consumption @ 22°C) (b–d), and in vitro mitochondrial production of reactive oxygen species (ROS) (e and f) in male mice. Error bars represent + standard error of the mean. N = 6 for each group, except where indicated differently. MR is expressed as kcal/wk per mouse (b), mL O2 · h–1 per g total body mass (c), and mL O2 · h–1 per g lean body mass (d). Arrows in (b) represent the different levels of dietary energy intake. In (e) and (f), numbers in each column represent sample size, and columns without a common letter above them are significantly different (p <.05)

 
Resting oxygen consumption at 22°C was measured in all mice on the day before they were killed for biochemical analysis. This metabolic rate was calculated as per whole mouse (Figure 2b), per g total body mass (Figure 2c), and per g lean body mass (Figure 2d). For easy comparison with their dietary energy intake (also shown in Figure 2b), the values per whole animal have been converted to kcal/wk, assuming 4.5 kcal/L O2 (26). As can be seen from this figure, the metabolic rate per whole mouse remained essentially constant throughout the 6 months of the experiment for the mice fed 125 kcal/wk. The average rate for these mice throughout this period was equivalent to approximately 80 kcal/wk, which represents ~64% of their energy intake. Mice provided with 85 kcal/wk had a resting metabolic rate equivalent to 62 kcal/wk after 1 month and an average of 56 kcal/wk following 3 and 6 months on this level of energy provision. These metabolic rates represent 73% and 65% of dietary energy intake, respectively. Mice provided with 50 kcal/wk had a metabolic rate equivalent to about 28 kcal/wk, 42 kcal/wk, and 72 kcal/wk, respectively, after 1, 3, and 6 months. These values (respectively) represent 56%, 84%, and 144% of weekly energy intake. The mice on the lowest energy provision (40 kcal/wk) had rates of oxygen consumption equivalent to 100%, 121%, and 178% of dietary energy intake after 1, 3, and 6 months, respectively. Values of resting metabolism that are higher than dietary energy intake (such as for the 40 kcal/wk mice at 3 and 6 months and for the 50 kcal/wk mice at 6 months) are of course impossible to sustain over an extended period if body mass is being maintained. They are likely artefacts in the comparison and represent elevated levels of oxygen consumption of these mice in the metabolic chamber, either due to activity of the mice or, most likely, due to thermoregulatory considerations. Oxygen consumption was measured at 22°C (the same temperature at which they were housed in the animal house); this temperature is below the thermoneutral zone of mice. In the metabolism chamber, this temperature will represent a more challenging cold environment than the mice would experience in their cages where they had the advantage of bedding material (which was absent in the metabolism chamber). Such thermoregulatory considerations will likely also be exacerbated by the lower insulation of these mice due to their relatively reduced skin and fur (see Figure 1g). It is of interest to note that, although daily activity (measured in the animal house) of the two energy-deprived groups decreased over the period of the experiment (Figure 2a), metabolic rate measured in the metabolism chamber increased over the same period (Figure 2b).

When metabolic rate was expressed on a mass-specific basis (Figure 2c and d), it can be seen that throughout the period of the experiment there was a decrease in mass-specific metabolic rate of the mice on the two high-energy diets but an increase in that of the mice on the two low-energy diets. This was true whether metabolic rate was expressed "per g total body mass" or "per g lean body mass." After 1 month on their respective diets, there were no significant differences between any of the four experimental treatments. After 6 months, however, the mice on the two low energy diets had significantly higher mass-specific metabolic rates than did the mice provided the two higher energy diets.

Mitochondrial ROS Production
The effects of CR for 3 and 6 months on the level of in vitro mitochondrial ROS production are illustrated in Figure 2e for liver and Figure 2f for skeletal muscle. As can be seen from both of these figures, the CR effect on mitochondrial in vitro ROS production was relatively small. For mitochondria from both tissues, there were no significant differences between any of the experimental treatments at 3 months. At 6 months there was no significant difference between any of the treatments for skeletal muscle mitochondria whereas, for liver mitochondria only, the highest and lowest energy provisions were significantly different from one another. Similarly, there was a significant increase for both liver and skeletal muscle mitochondria between 3 and 6 months for the mice on 125 kcal/wk but not for any other experimental treatments.

Membrane Fatty Acid Composition
Tables 2–8 provide the percent composition of phospholipids from liver (Table 2), kidney (Table 3), brain (Table 4), heart (Table 5), and skeletal muscle (Table 6), and from liver mitochondria (Table 7) and skeletal muscle mitochondria (Table 8). Using the fatty acid composition of each sample and the relative peroxidative susceptibility of each individual fatty acid, it is possible to calculate a PI for the phospholipids of each sample (see Methods). This value indicating the relative susceptibility of membrane phospholipids to peroxidative damage is presented in Figure 3 for liver (a), heart (b), kidney (c), brain (d), liver mitochondria (e), and skeletal muscle mitochondria (f).


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  		Table 2. Effect of Level and Duration of Dietary Calorie Restriction in Mice on Fatty Acid Composition of Liver Phospholipids.

 

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  		Table 3. Effect of Calorie Restriction on Fatty Acid Composition of Kidney Phospholipids.

 

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  		Table 4. Effect of Calorie Restriction on Fatty Acid Composition of Brain Phospholipids.

 

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  		Table 5. Effect of Calorie Restriction on Fatty Acid Composition of Heart Phospholipids.

 

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  		Table 6. Effect of Calorie Restriction on Fatty Acid Composition of Skeletal Muscle Phospholipids.

 

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  		Table 7. Effect of Calorie Restriction on Fatty Acid Composition of Liver Mitochondrial Phospholipids.

 

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  		Table 8. Effect of Calorie Restriction on Fatty Acid Composition of Skeletal Muscle Mitochondrial Phospholipids.

 

Figure 03
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  		Figure 3. Effect of four levels of energy intake (125, 85, 50, and 40 kcal/wk) on the peroxidation index of phospholipids from liver (a), heart (b), kidneys (c), brain (d), liver mitochondria (e), and skeletal muscle mitochondria (f) from male mice. Peroxidation index is a measure of susceptibility to lipid peroxidation (see Methods for calculation). Error bars represent ± standard error of the mean. Sample size for each data point is shown in Tables 2–8

 
As can be seen in Figure 3, CR resulted in significant changes in the fatty acid composition of phospholipids (and thus membranes) of liver, kidney, brain, and heart, such that the PI was significantly decreased. The statistical significance of these changes was greatest in liver, kidney, and heart, and was less pronounced in the brain. In the skeletal muscle phospholipids (results not shown in Figure 3), there was no significant effect of dietary energy intake. For the mitochondrial phospholipids from liver there was a highly significant CR effect on the PI, and for skeletal muscle mitochondrial phospholipids, the CR effect was very close to statistical significance (p =.053).

Although the CR effects varied between the tissue phospholipids and between mitochondrial phospholipids, the pattern of the response was relatively similar. The highest PI values were observed in the mice receiving 125 kcal/wk, whereas the lowest values were found for the mice receiving the two lowest dietary energy intakes, with the values for the 85 kcal/wk mice being intermediate. For the mice on the highest energy intake (125 kcal/wk) the PI was either relatively constant with time or showed an increase (especially after 6 months). The reduction in PI with lower dietary energy provision was manifest within 1 month on the respective diet, and the relative difference (compared to the 125 kcal/wk mice) did not change much with time.

Examination of Tables 2–8 shows that the reduction in the peroxidative susceptibility of membrane lipids following CR is due to changes in the types of unsaturated fatty acids (not due to a reduction in the total percentage of unsaturated fatty acids in membranes). The changes observed in mitochondrial phospholipids were essentially the same as those observed for the total phospholipids of the tissue from which the mitochondria were prepared. In liver and kidney, CR resulted in an increase in the relative content of monounsaturates, a decrease in the relative content of omega-3 PUFA (particularly 22:6), and no change in the total omega-6 PUFA. However, within the omega-6 PUFA, there was a decrease in the relative amount of arachidonic acid (20:4n-6) and an increase in linoleic acid (18:2n-6). In the brain there was a small decrease in 22:6n-3 content. CR resulted in a statistically significant (p <.0001) reduction in the ratio of 20:4n-6/18:2n-6 within the n-6 PUFA in the brain, liver, kidney, skeletal muscle, and liver mitochondria (results not shown). In the heart phospholipids, there was a small increase in n-6 PUFA (that was due to increases in 18:2n-6 content) with CR.


   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The finding that blood hemoglobin levels, after 3 months, were not different between treatment groups is indicative that CR mice were subject to undernutrition and not malnutrition. At the beginning of the CR regimen, there was a 3-fold range in the mass-specific energy intake of the mice (i.e., 125 kcal/wk compared to 40 kcal/wk); however, after 6 months, body size of the mice had changed such that there was essentially no difference in mass-specific food energy intake between the four experimental groups. At the end of the study, all mice were receiving between 1.9 and 2.2 kcal/wk per g total body mass. As outlined in the title of the article by McCay and colleagues (27) originally describing the effects of CR in rats, one of its major effects is the determination of ultimate body size.

Other researchers have previously examined CR, and our findings are consistent with past findings. The mice provided with the high-energy provision accumulated more body fat than did those on the low-energy intake. After 6 months, the mice on 125 kcal/wk had body fat stores equivalent to 155% of their weekly energy provision (i.e., ~11 days worth of food), those provided with 85 kcal/wk had 114% (i.e., 8 days), while the 50 kcal/wk mice had body fat equivalent to 28% of weekly energy intake (i.e., ~2 days) and the 40 kcal/wk mice had the equivalent of 16% of their weekly energy intake (i.e., ~1 day). The size of internal organs decreased with CR which is consistent with past findings (12,17,28,29) as did lean body mass, such that for some internal organs there was no change in size relative to lean body mass.

With the "live fast–die young" perspective inherent in the rate-of-living theory of aging, many investigators initially thought that the life span–extending properties of CR were due to it reducing the mass-specific rate of metabolism. It was shown some time ago, however, that CR in rats does not reduce their mass-specific metabolic rate, expressed relative to either total body mass or lean body mass (30). We have observed the same situation here in mice. Although resting metabolic rate per whole animal is reduced in CR mice, the reduction in body mass due to CR is such that there was no reduction in mass-specific metabolic rate whether expressed relative to total body mass or lean body mass. Indeed, in CR mice, mass-specific metabolic rate appeared to increase over the 6 months of the experiment. We are unsure of the reason for this increase, but it is of interest that it has been recently reported that the energy expenditure of CR rats is higher than predicted from their altered body composition (31). Not only did CR not reduce resting metabolic rate but it also did not reduce daily activity and thus presumably daily energy expenditure. This lack of a decrease in mass-specific metabolism during CR would appear to be a general phenomenon in that dietary restriction in both wild-type and the long-living CHICO-mutant strain of D. melanogaster also did not result in a change in mass-specific rate of metabolism (32). It is clear that the life-span extension due to CR is not due to a decrease in the mass-specific rate-of-living.

CR is associated with lower levels of oxidative damage, and this is thought to cause its life-span extension effect (1,2). The reason for low levels of oxidative damage is not completely understood. Measurement of mitochondrial in vitro production of superoxide, hydrogen peroxide, or both (referred to as ROS production) by a number of investigators shows that it is lowered during long-term CR, whereas results are more equivocal after short-term CR (33). Here we observed only a very small CR effect on such in vitro ROS production for liver and skeletal muscle mitochondria from mice and only after 6 months of CR. Whereas 1 year of CR lowered in vitro ROS production by heart and liver mitochondria of rats (34,35), 6 weeks of CR resulted in no effect on heart mitochondria (34) but a decrease in liver mitochondria (35). Other studies, however, have shown no effect on in vitro H2O2 production by isolated liver mitochondria after either 1 or 6 months (12), or after 12 months CR (36) in rats. One exception to the lack of an effect by short-term CR is the report that in vitro H2O2 production by isolated muscle mitochondria was reduced after both 2 weeks and 2 months of CR in the rat (37). A separate study, however, showed no effect of 2 months CR on rat skeletal muscle mitochondria (38). It has been recently reported that 4-month CR in rats had no effect on ROS production in intact hepatocytes (39) despite effects reported for isolated liver mitochondria (33). This raises the perennial biochemical problem of relating the in vitro findings for isolated mitochondria to both the situation of mitochondria within isolated cells and the in vivo situation.

Although superoxide and hydrogen peroxide are important ROS, they are not the only (nor even necessarily the majority of) molecules that can be described as ROS. Many of the products of lipid peroxidation are themselves powerful and important ROS (40). It is for this reason that lipid peroxidation is an autocatalytic process and that the fatty acid composition of membranes is likely an important component of the aging process (14–16). Fatty acids differ in their susceptibility to peroxidative damage because it is the single-bonded carbons residing between the doubled-bonded carbons in fatty acyl chains that are attacked by ROS (40). The more double-bonds in an individual acyl chain, the more prone it is to peroxidation and consequent production of ROS. Knowing the fatty acid composition of a membrane bilayer makes it possible to calculate an index of its susceptibility to peroxidation which can be expressed quantitatively as a PI value (see end of Methods section). It is sometimes mistakenly suggested that it is the total number of double bonds in a membrane bilayer (i.e., the UI) that determines its susceptibility to oxidative damage. This not necessarily the case, however, and an extreme example can illustrate the situation. A membrane bilayer containing only monounsaturated acyl chains will have a UI = 100 and a PI = 2.5, whereas a bilayer containing 95% saturated acyl chains and 5% docosahexaenoic acid will have a UI = 30 and a PI = 40. Thus the bilayer with the 5% highly polyunsaturated docosahexaenoic acid will have less than one-third the number of double bonds but will be 16 times more susceptible to lipid peroxidation than the completely monounsaturated bilayer.

In an important article, Laganiere and Yu (7) first showed that CR of rats changed the fatty acid composition of liver mitochondrial and microsomal membranes such that they became less susceptible (and thus more resistant) to lipid peroxidation. Since this seminal observation, other studies have reported similar CR-induced changes for membranes of other tissues (9–11,41,42), as well as for different classes of liver phospholipids (43,44). All of these studies involved long-term CR in rats, with the shortest CR period examined being 10 weeks (43). Here we report a similar CR-induced decrease in the peroxidation susceptibility of membrane lipids from a variety of tissues in mice. In general, these changes vary with the degree of CR. They are also early CR effects, being manifest within 1 month (which was the earliest sampling period). Indeed, a surprising finding was that, in general, the differences manifested after 1 month of CR were quantitatively similar to those observed after both 3 and 6 months CR.

The decrease in membrane susceptibility to peroxidation varied in degree between tissues but was due to similar changes in the different tissues. There was a decrease in the relative content of n-3 PUFA in almost all tissues largely due to decreases in the highly polyunsaturated docosahexaenoic acid (22:6n-3). This decrease in n-3 PUFA was compensated by an increase in the relative content of peroxidation-resistant monounsaturated fatty acids in liver, kidney, heart, and brain phospholipids. Although the total content of n-6 PUFA did not tend to change with CR, there was a decrease in most tissues in peroxidation-prone arachidonic acid (20:4n-6) which was compensated by an increase in the relative content of the more peroxidation-resistant linoleic acid (18:2n-6). Such compensatory increases in peroxidation-resistant fatty acids are illustrated by the fact that, although there was a very significant (p <.0001) diet effect on liver mitochondrial PI (Figure 3e), the diet effects on liver mitochondrial UI were not statistically significant (Table 7).

In all tissues examined, we have measured the composition of total phospholipids, which obviously combines all subcellular membranes in one measurement. In liver, where mitochondrial phospholipids were separately measured, the changes were essentially the same in both the total phospholipids and in mitochondrial phospholipids.

Skeletal muscle phospholipids were the most refractory to change, showing negligible changes in fatty acid composition following CR. As with the other tissues, analyzing the total tissue phospholipids pooled all subcellular membranes. It may be that changes in one direction in a particular subcellular compartment were offset by changes in the other direction in a different subcellular membrane such that there is no observed change in the total tissue phospholipids. There is some evidence that this may be the case. Whereas mitochondrial phospholipids from skeletal muscle showed small decreases in 22:6n-3 with CR, total phospholipids showed either a small increase or no significant change. For example, following 1-month CR there was a decrease in the ratio of 20:4n-6 to 18:2n-6 in total phospholipids from skeletal muscle, similar to that in other tissues, but no decrease in PI because of a compensating increase in 22:6n-3 content. Cefalu and colleagues (10) have also reported increased 22:6n-3 content of rat skeletal muscle membranes with CR and related it to increased insulin sensitivity of peripheral tissues during CR in rats.

Some recent studies (11–13) have failed to show the effects of CR on membrane composition observed here and elsewhere; however, it has been the fatty acid composition of mitochondrial total lipids rather than mitochondrial phospholipids that has been measured in these studies. Thus the measurements will have also included the fatty acid composition of any cellular triglycerides associated with the mitochondrial pellet in their reported values. In view of the fact that CR will likely have a significant influence on the triglyceride content of cells, and that triglycerides are often less unsaturated than are phospholipids, it is possible that CR effects on the fatty acid composition of these mitochondrial membranes have been masked in these studies.

The changes in membrane composition are early biochemical effects of CR and are possibly brought about by even earlier CR-induced hormonal changes. Insulin, triiodothyronine, and growth hormone are all significantly and rapidly lowered by CR (1) and are the likely candidates for instigating CR-induced changes in membrane fatty acid composition. The low insulin levels of diabetes are associated with low 20:4n-6 and high levels of 18:2n-6 in membrane lipids (45–47), and CR-induced changes in liver mitochondrial function in rats can be reversed by insulin (48). It has been shown recently that insulin and growth hormone increase the DBI (double bond index, which is related to but not the same as the PI; see earlier discussion) of liver phospholipids of rats during both ad libitum feeding and CR (49). Similarly, many studies have shown that hypothyroidism affects the fatty acid composition of membrane lipids, with the predominant change being the same as that observed during CR, namely, a decrease in the 20:4n-6 and an increase in 18:2n-6 (50). These thyroid-induced changes have been observed for a wide variety of tissues and subcellular membranes, however, two notable exceptions are the sarcoplasmic reticulum and sarcolemmal membranes of muscle (50).

Changes in the fatty acid composition of membranes will have effects on the rate of lipid peroxidation. Ethane is a volatile end product of the peroxidation of n-3 PUFA, whereas pentane is a volatile product from the peroxidation of n-6 PUFA; the exhalation rates of both have been used as indicators of in vivo lipid peroxidation (51). In one study, the age-associated increase in pentane exhalation of rats was suppressed by long-term CR, but there was no effect of CR on the ethane exhalation of rats (52). In another study, however, CR for 2 weeks decreased ethane exhalation of rats by 33% (53). Whether such a decrease is due to changes in membrane fatty acid composition or metabolic rate (or both) is not known but is worthy of further investigation.

Because many of the products of lipid peroxidation are potent ROS, changes in the fatty acid composition of membranes can also affect oxidative damage to other important cellular constituents. For example, increasing the PI of membrane lipids of rats by manipulation of their diet also resulted in an increase in oxidative damage to proteins and mitochondrial DNA (but not to nuclear DNA) in their brain and liver (54). The reduced oxidative damage so often observed with CR (1) may be primarily due not to changes in the mitochondrial production of superoxide and hydrogen peroxide, but rather to a decrease in the ROS produced by lipid peroxidation secondary to hormone-mediated changes in the fatty acid composition of membranes.

The importance of the fatty acid composition in understanding aging has been recently discussed (14,16), and the PI of liver mitochondrial phospholipids of different mammal and bird species has been shown to be inversely related to maximum life span, such that mammals and birds exhibit the same relationship. This relationship showed that a 24% decrease in the PI (and thus peroxidation susceptibility) of liver mitochondrial membrane lipids was associated with a doubling of life span (16). It has been speculated that one of the mechanisms by which CR extends life span is by lowering the PI of membrane phospholipids [e.g., (14)]. Our results are in agreement with such speculation.


   Acknowledgments
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 Abstract
 Methods
 Results
 Discussion
 References
 
Funding was provided by grants from the University of Wollongong and the Australian Research Council.

We thank Dr. Todd Mitchell for technical assistance, as well as Belinda Ferrone and Amanda Lane for assistance with the fatty acid analysis.

Nigel Turner is now with the Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.


   Footnotes
Top
 Abstract
 Methods
 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received November 27, 2005

Accepted February 16, 2006


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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